Work from our lab and others has established that expression of the p16INK4a tumor suppressor mechanism plays a critical role in mammalian aging. The expression of p16INK4a in the setting of certain cellular stresses and tissue pathology clearly plays a beneficial role in limiting important age-associated conditions associated with excess proliferation such as cancer and atherosclerosis. The activation of p16INK4a throughout life, however, is associated with the accumulation of cells that have undergone senescence, a permanent form of growth arrest, which is also associated with the elaboration of pathogenic, pro-inflammatory cytokines. Therefore, the beneficial cancer-preventing expression of p16INK4a compromises some aspects of organismal fitness with aging by limiting the regeneration and repair of certain self-renewing compartments. During the prior ten years of this proposal, our group, with collaborators, has provided significant evidence for this model. In 2004, we showed that p16INK4a is a faithful biomarker of mammalian aging. In 2006, we showed that p16INK4a-deficient animals demonstrate a resistance to some aging phenotypes in pancreatic -cells, neural stem cells and hematopoietic stem cells, while transgenic animals expressing excess p16INK4a demonstrate an accelerated functional decline with aging in these compartments. In 2009, we developed an approach to measuring p16INK4a expression with aging in humans, and employed this assay to show the age-promoting effects of tobacco use, physical inactivity, chronic HIV infection, and cytotoxic chemotherapy in people. We also identified a highly common polymorphism in humans that strongly affects p16INK4a expression and is associated with atherosclerotic disease. In 2011, we showed an important effect of p16INK4a expression on the physiology of aging B- vs. T-lymphocytes, and provided an explanation for how p16INK4a expression limits atherosclerotic disease. Finally, in 2013, we reported in Cell that p16INK4a expression with aging does not predict malignancy risk in mice, and showed that locus activation results in vivo from cell-non-autonomous mechanisms. In the renewal of this proposal, we seek to extend these prior observations to further enhance our understanding of how p16INK4a influences mammalian aging. In specific aim I, we employ novel reporter and p16CRE alleles to study whether p16INK4a expressing cells are always senescent in vivo. In specific aim II, we will examine the effects of depleting p16INK4a-expressing cells on tumorigenesis and aging using a genetic or immune approach. In specific aim III, we will examine the effects of alterations in the cellular epigenetic state on expression f p16INK4a and somatic stem cell function with aging. Through these approaches, we will further delineate the contribution of p16INK4a expression and cellular senescence to mammalian aging.